Several studies suggest that B cells contribute to kidney allograft rejection. B cell infiltrates in the graft are often observed during rejection and have been associated with poor outcome. Gene expression studies have also revealed B cell transcripts in graft tissue during steroid refractory rejection. The exact phenotype, antigenic specificity and function of infiltrating B cells, however, are still unknown. In preliminary studies, we isolated mononuclear cells from kidney tissue following transplantectomy for rejection. Phenotypic experiments demonstrated that graft infiltrates were enriched in CD27+ class-switched B cells compared to the blood. We subsequently immortalized B cell lines from these specimens and assessed their specificity using the antibodies in the culture supernatant. A significant fraction of them appeared to recognize HLA molecules, autoantigens and apoptotic cells. We hypothesize that these polyreactive B cells are primarily involved in the uptake and presentation of antigens to T cells either directly or through the antibodies they secrete. Aim-1. To assess the differentiation stage, expansion and specificity of graft infiltrating B cells: We will test our hypothesis that B cells are activated and differentiate directly in rejected graft tissue. First, the distribution and phenotype of B cells within the rejected graft tissue will be assessed in biopsy and nephrectomy samples. Then, analyses of the Ig heavy chain CDR3 sequences in early biopsy samples, nephrectomy specimens as well as contemporary blood samples will identify expanded B cell clones in early or late rejection. In parallel, B cells isolated from explanted grafts will be immortalized and cultured in vitro. Selected clones corresponding to B cells expanded in situ will be further analyzed. Aim-2. To determine the functional properties of antibodies secreted by graft infiltrating B cells: Monoclonal antibodies secreted by immortalized graft infiltrating B cells will be assessed primarily for their capacity to contribute to antigen uptake and presentation to T cells. In particular, we will examine the antibodies[unreadable] capacity to bind to released antigen or apoptotic cells and enhance their uptake by antigen presenting cells (APC). Additional pathogenic functions of antibodies will also be examined including their capacity to fix and activate complement, leading to the deposition of C4d. Aim-3. To define the function of graft infiltrating B cells: Aside from their capacity to secrete pathogenic antibodies, we will examine whether graft infiltrating B cells can also directly contribute to the rejection process, primarily through their capacity to uptake and present antigens to T cells. More specifically, we will assess the capacity of B cells showing specificity to apoptotic cells, to [unreadable]scavenge[unreadable] apoptotic bodies and present antigens included in the bodies to T cells. Other possible functions of infiltrating B cells will also be examined.